Methods and intermediates for preparing macrolactams

ABSTRACT

The present invention includes compounds useful as intermediates in the preparation of macrolactams, methods for preparing the intermediates, and methods for preparing macrolactams. One use of the methods and intermediates described herein is in the production of macrolactam compounds able to inhibit HCV NS3 protease activity. HCV NS3 inhibitory compounds have therapeutic and research applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage application of International PatentApplication No. PCT/US2014/060348, filed Oct. 14, 2014, which claimspriority to U.S. Provisional Patent Application No. 61/927,701, filedJan. 15, 2014,and U.S. Provisional Patent Application No. 61/892,790,filed Oct. 18, 2013.

FIELD OF THE INVENTION

The present invention relates to process and intermediates that can beused for preparing macrolactams. One use of the methods andintermediates described herein is the production of macrolactamcompounds able to inhibit HCV NS3 protease activity. HCV NS3 inhibitorycompounds have therapeutic and research applications.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) infection is a major health problem. HCVinfection leads to chronic liver disease, such as cirrhosis andhepatocellular carcinoma, in a substantial number of infectedindividuals.

Several virally-encoded enzymes are putative targets for therapeuticintervention, including a metalloprotease (NS2-3), a serine protease(NS3), a helicase (NS3), and an RNA-dependent RNA polymerase (NS5B). TheNS3 protease is located in the N-terminal domain of the NS3 protein.NS4A provides a cofactor for NS3 activity.

Examples of publications describing macrolactam compounds able toinhibit HCV protease activity include: Harper et al., WO 2010/011566;Liverton et al., WO 2009/134624; McCauley et al., WO 2009/108507;Liverton et al., WO 2009/010804; Liverton et al., WO 2008/057209;Liverton et al., WO 2008/051477; Liverton et al., WO 2008/051514;Liverton et al., WO 2008/057208; Crescenzi et al., WO 2007/148135; DiFrancesco et al., WO 2007/131966; Holloway et al., WO 2007/015855;Holloway et al., WO 2007/015787; Holloway et al., WO 2007/016441;Holloway et al., WO 2006/119061; Liverton et al., J. Am. Chem. Soc.130:4607-4609, 2008; and Liverton et al., Antimicrobial Agents andChemotherapy 54:305-311, 2010.

SUMMARY OF THE INVENTION

The present disclosure provides methods and intermediates for preparingmacrolactams. One use of the methods and intermediates described hereinis in the production of macrolactam compounds able to inhibit HCV NS3protease activity. HCV NS3 inhibitory compounds have therapeutic andresearch applications.

In particular, the present disclosure provides a method of preparing acompound of Formula C:

wherein n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6,7, and 8; X¹ and X² are each independently selected from the groupconsisting of Br, Cl, and I; and R⁵ is selected from the groupconsisting of C₁₋₈ alkyl, C₃₋₈ cycloalkyl, aryl, and heteroaryl groupsand R⁵ is substituted by 0, 1, 2, 3, or 4 substituents independentlyselected from the group consisting of C₁₋₆ alkyl, —C₂₋₆ alkenyl,—C₂₋₆alkynyl, aryl, halogen, —NH₂ and —OH. The method comprises the steps of(1) reacting

where LG is selected from the group consisting of halogen atoms,—O—SO₂R⁸, —O—PO(OR⁸)₂ or a protecting group and each R⁸ is independentlyselected from the group consisting of C₁₋₈ alkyl, C₃₋₈ cycloalkyl, aryl,and heteroaryl groups and each R⁸ is independently substituted by 0, 1,2, 3₅ or 4 substituents independently selected from the group consistingof C₁₋₆ alkyl, —C₂₋₆alkenyl, alkynyl, aryl, halogen, —NH₂; and —OH, andthe protecting group is selected from —OSiR⁸ and —OR⁸, with a chiralalcohol and

to produce

where each R¹ is independently selected from the group consisting ofC₁₋₈ alkyl, aryl, and heteroaryl groups, or two R¹ are taken, togetherwith the O—P—O atoms to which they are attached, to form a ringcontaining 5-19 atoms; and where R² and R³ are each selected from thegroup consisting of H, C₁₋₈ alkyl, and —O—C₁₋₈ alkyl groups, or where R²is H, and R³ is a —O—C₁₋₈ alkyl group, or R² and R³ are each H or C₁₋₈alkyl groups or are taken together with the nitrogen atom to which theyare attached to form a ring containing 5-19 atoms; (2)reacting

with a Grignard reagent to produce

where R⁴ is selected from the group consisting of C₁₋₈ alkyl,substituted C₁₋₈ alkyl, aryl, substituted aryl, heteroaryl, andsubstituted heteroaryl groups and R⁴ is substituted by 0, 1, 2, 3, or 4substituents independently selected from the group consisting of C₁₋₆alkyl, C₂₋₆, alkenyl, C₂₋₆ alkynyl, aryl, halogen, —NH₂ and —OH; (3)halogenating

to produce

where X¹ and X² are each independently selected from the groupconsisting of Br, Cl, and I; and (4) oxidizing

to produce oxygen-inserted compounds

where R⁵ is selected from the group consisting of C₁₋₈ alkyl, C₃₋₈cycloalkyl, aryl, and heteroaryl groups and R⁵ is substituted by 0, 1,2, 3 or 4 substituents independently selected from the group consistingof C₁₋₆ alkyl, C₁₋₆ alkenyl, C₁₋₆ alkynyl, aryl, halogen, —NH₂ and —OH.

In addition, the present disclosure provides a method of preparing acompound of Formula B:

or a salt thereof, wherein n, R⁷, and R⁶ are as described above. Themethod comprises preparing a compound of Formula C according to themethod of claim 1; and converting

or a salt thereof. The compounds of Formula B may be prepared by makingcompounds of Formula C and converting the compounds of C into compoundsof Formula B.

Other embodiments, aspects and features of the present invention areeither further described in or will be apparent from the ensuingdescription, examples and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a characteristic X-ray diffraction pattern for thecrystalline tert-butylamine salt of the alkyne acid of Example 10.

FIG. 2 provides a characteristic X-ray diffraction pattern for themethanesulfonate salt of(2S,4R)-4-(3-chloro-7-methoxyquinoxalin-2-yloxy)-2-(methoxycarbonyl)pyrrolidineof Example 13

FIG. 3 provides a characteristic X-ray diffraction pattern for thecrystalline macrocyclic alkyne ester anhydrous form I of Example 14A.

FIG. 4 provides a characteristic ¹³C NMR spectrum for the crystallinemacrocyclic alkyne ester anhydrous form I of Example 14A.

FIG. 5 provides a typical differential scanning calorimetry (DSC) curveof the crystalline macrocyclic alkyne ester anhydrous form I of Example14A.

FIG. 6 provides a characteristic X-ray diffraction pattern for thecrystalline macrocyclic ester anhydrous form II of Example 14B.

FIG. 7 provides a characteristic ¹³C NMR spectrum for the crystallinemacrocyclic alkyne ester anhydrous form II of Example 14B.

FIG. 8 provides a typical differential scanning calorimetry (DSC) curveof the crystalline macrocyclic alkyne ester anhydrous form II of Example14B.

FIG. 9 provides a characteristic X-ray diffraction pattern of thecrystalline IPA solvate/hydrate of Example 14C.

FIG. 10 provides a characteristic ¹³C NMR spectrum for the crystallinemacrocyclic alkyne ester anhydrous form II of Example 14C.

FIG. 11 provides a typical differential scanning calorimetry (DSC) curveof the crystalline IPA/water mixed solvate/hydrate of Example 14C.

DETAILED DESCRIPTION OF THE INVENTION

Macrolactam compounds able to inhibit HCV activity have different usesincluding inhibiting HCV activity in vivo, inhibiting HCV activity invitro, and inhibiting HCV NS3 enzymatic activity. In vivo inhibition ofHCV activity can be used for therapeutic applications. Inhibiting HCVactivity in vitro has different applications including being used toobtain HCV resistant mutants, further characterizing the ability of afunctional group to inhibit HCV replicon or enzymatic activity, andstudying HCV replication or protease activity.

The methods and intermediates described herein can be used to synthesizemacrolactams, such as Compound A and compounds varying from Compound Aby one or more functional group. Compound A has the following structure:

Functional groups that can be modified include a different heterocyclegroup, a different alkyl in place of the tert-butyl group, andalteration of the cyclopropylsulfonyl functional group and thecyclopropyl amide moiety (e.g., with an ethyl group replacing theethylene and/or a methylcyclopropyl group replacing the cyclopropylgroup).

Different intermediates and synthesis protocols are illustrated hereinwhere Compound A was ultimately obtained. However, it is understood thatbased on the guidance provided herein other macrolactams can be producedusing appropriate intermediates and by adding or modifying differentfunctional groups. Examples of different macrolactams having differentfunctional groups are provided in McCauley et al., WO 2011/014487;Harper et al., WO 2010/011566; Liverton et al., WO 2009/134624; McCauleyet al., WO 2009/108507; Liverton et al., WO 2009/010804; Liverton etal., WO 2008/057209; Liverton et al., WO 2008/051477; Liverton et al.,WO 2008/051514; Liverton et al., WO 2008/057208; Crescenzi et al., WO2007/148135; Di Francesco et al., WO 2007/131966; Holloway et al., WO2007/015855; Holloway et al., WO 2007/015787; Holloway et al., WO2007/016441; Holloway et al., WO 2006/119061; Liverton et al., J. Am.Chem. Soc., 130:4607-4609, 2008; and Liverton et al., AntimicrobialAgents and Chemotherapy 54:305-311, 2010.

Harper et al., WO 2010/011566 describes an alternative method for makingCompound A. Harper et al., WO 2010/011566, also includes dataillustrating the ability of Compound A to inhibit HCV replicon activityand NS3/4A. In addition, Yasuda et al., WO 2013/028471 and Xu et al., WO2013/028470 describe methods and intermediates for making Compound A,and Beutner et al., WO 2013/028465 describes crystal forms of CompoundA.

Intermediates and procedures that can be used to produce macrolactamscan be illustrated by taking into account: (1) cyclopropyl linkersynthesis: (2) heterocycle synthesis; and (3) forming a macrolactamusing the cyclopropyl linker and heterocycle group, and optionallyadding or modifying different functional groups. The optionally addedfunctional groups can be used to provide for, or enhance, the ability ofa compound to inhibit HCV NS3 activity and/or HCV replication.

Cyclopropyl Linker Synthesis

Cyclopropyl linker intermediates useful for preparing Compound A andanalogues thereof include compounds of Formula B,

wherein n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6,7 and 8; and R⁷ is selected from the group consisting of acetyl and

in which R⁶ is selected from the group consisting of C₁₋₈ alkyl, C₃₋₈cycloalkyl, aryl and heterocycle groups. In embodiments, the compound ofFormula B is a compound in which R⁷ is acetyl. In separate embodiments,the compound of Formula B is a compound in which R⁷ is

and R⁶ is selected from the group consisting of C₂-C₆ alkyl groups. Inparticular embodiments, the compound of Formula B is a compound in whichR⁷ is

and R⁶ is tert-butyl; that is, the compound of Formula B is(S)-3,3-dimethyl-2-(((1R,2R)-2-(pent-4-ynyl)cyclopropoxy) carbonylamino)butonic acid, Compound B1:

Scheme A illustrates an overall scheme that can be used to preparecompounds of Formula B, particularly Compound B1, and differentintermediates. Each of the individual steps of Scheme A provides forembodiments, and combinations of steps together provide additionalembodiments. Further embodiments include steps upstream and downstreamfrom a particular step, such as those illustrated by the Examples.Unless specifically indicated, any variable maintains its definition asprovided in an earlier structure when that variable is used in a laterstructure.

In the compounds and intermediates of Scheme A, n is 0, 1, 2, 3, 4, 5,6, 7 or 8.

In the compounds and intermediates of Scheme A,

where LG is selected from the group consisting of halogen atoms,—O—SO₂R⁸, —O—PO(OR⁸)₂, and a protecting group, and each R⁸ isindependently selected from the group consisting of C₁₋₈ alkyl, C₃₋₈cycloalkyl, aryl, and heteroaryl groups, and the protecting group isselected from —OSiR and —OR⁸, is prepared by (i) reacting

with a magnesium source to produce

and (ii) reacting

with

to produce

In the compounds and intermediates of Scheme A, X is selected from thegroup consisting of halogen atoms. Preferably, X is selected from thegroup consisting of Br, Cl, and F. In specific embodiments, X is Br.

In the compounds and intermediates of Scheme A, LG is a leaving groupselected from the group consisting of halogen atoms, —O—S₂R⁸,—O—PO(OR⁸)₂ and a protecting group, where each R⁸ is independentlyselected from the group consisting of C₁-C₈ alkyl, C₃-C₈ cycloalkyl,aryl and heteroaryl groups, and the protecting group is selected from—O—SiR⁸ and —O—R⁸. In embodiments, LG is a leaving group selected fromthe group consisting of halogens, mesylate

—O—PO(OR⁸)₂, tosylate

—OCH₂OCH₃ and —OSiR⁸ ₃, where each R⁸ is independently selected from thegroup consisting of C₁-C₈ alkyl. It will be understood that a protectinggroup may be converted to an appropriate leaving group, directly orindirectly (such as by stepwise conversion), to achieve the desireddownstream chemistry. In specific embodiments, LG is selected from thegroup consisting of Br, Cl, F, mesylate and tosylate. In particularembodiments, LG is Cl.

In the compounds and intermediates of Scheme A, PG is a leaving group topromote carbamate bond formation via coupling alcohol intermediate withan amino acid. PG may be selected from imidazoyl and succinimidyl. Thecoupling may be carried out, in embodiments, with promoters oradditives, such as 2-hydroxypyridine-N-oxide (HOPO), hydroxylsuccinimide (HOSu), imidazole and imidazole HCl salt.

In embodiments, the third step illustrated in Scheme A employs a chiralalcohol, such as chlorohydrin.

In the compounds and intermediates of Scheme A, each R¹ is independentlyselected from the group consisting of C₁-C₈ alkyl, aryl and heteroarylgroups. In embodiments, the two R¹ are taken, together with the O—P—Oatoms to which they are attached, to form a ring. In particularembodiments, each R¹ is independently selected from the group consistingof C₁-C₈ alkyl groups. In specific embodiments, each R¹ is independentlyethyl.

In the compounds and intermediates of Scheme A, R² and R³ are eachselected from the group consisting of H, —O—C₁-C₈ alkyl and C₁-C₈ alkylgroups. In embodiments, R² is H, and R³ is —O—C₁-C₈ alkyl, or R² and R³are H or C₁-C₈ alkyl and are taken together with the nitrogen atom towhich they are attached to form a ring. In particular embodiments, R²and R³ are each methyl.

In particular embodiments, R¹ is independently ethyl and R² and R³ areeach methyl, such that

In the compounds and intermediates of Scheme A, R⁴ is selected from thegroup consisting of H, C₁-C₈ alkyl, substituted C₁-C₈ alkyl, aryl,substituted aryl, heteroaryl and substituted heteroaryl groups.

In the compounds and intermediates of Scheme A, X¹ and X² are eachindependently selected from the group consisting of Br, Cl and I.

In the compounds and intermediates of Scheme A, R⁵ and R⁶ areindependently selected from the group consisting of H, C₁-C₈ alkyl andC₃-C₈ cycloalkyl groups. In embodiments, R⁵ and R⁶ are independentlyselected from the group consisting of C₂-C₆ alkyl groups. In particularembodiments, R⁵ is methyl. In particular embodiments R⁶ is tert-butyl.

In embodiments, the step of converting

(compounds of Formula C) to

or a salt thereof, is accomplished by the steps of (i) de-halogenating

to produce

(ii) reacting

with a reagent containing a leaving group to produce

where LG is a leaving group; and (iii) reacting

with a reagent selected from carboimide groups and

to produce

and (iv) optionally forming a salt of

In some embodiments, the product compound is converted to apharmaceutically acceptable salt, such as a tert-butylamine salt,dibenzylamine salt or dicyclohexyl amine salt.

Scheme B illustrates an overall scheme that can be used to prepareCompound B1 and different intermediates. Each of the individual steps ofScheme B provides for embodiments, and combinations of steps togetherprovide additional embodiments. Further embodiments include stepsupstream and downstream from a particular step, such as thoseillustrated by the Examples.

In particular embodiments, the method of Scheme B comprises the steps of(1) reacting

with a magnesium source to produce

(2) reacting

with

to produce

(3) reacting

with chlorohydrin and

to produce

(4) reacting

with a Grignard reagent to produce

(5) brominating

to produce

(6) oxidizing

to produce

(7) de-brominating

to produce

(8) reacting

with

to produce

and (9) optionally forming a salt of

The compounds in Scheme A and Scheme B are in the neutral form unlessotherwise indicated.

Potential advantages of performing the different steps illustrated inScheme A and Scheme B, compared with a method of producing analternative cyclopropyl linker having an ethylene group, described inHarper et al., WO 2010/011566, include the possibility of increasedefficiency and cost-effectiveness, as well as the possibility of higheroverall yields when compared to prior processes, including the methodsdescribed in Yasuda et al., WO 2013/028471, and Xu et al., WO2013/028470. The process provided herein is an asymmetric synthesis,which applies a novel cyclopropanation reaction with a chiralchlorohydrin. Optically pure cyclopropanol intermediates are obtainedthrough a Baeyer-Villiger oxidation. In contrast, the previouslydescribed processes are racemic synthesis processes, which require useof selective enzymatic hydrolysis of racemic intermediates.

Carbamate bonds may be formed by use of coupling reagents, such asN,N′-disuccinmidyl carbonate (DSC) and carbonyl diimidazole (CDI) andthe like, as previously described, such as in Yasuda et al., WO2013/028471, and Xu et al., WO 2013/028470. The introduction ofpromoters or additives, such as 2-hydroxypyridine-N-oxide (HOPO),hydroxyl succinimide (HOSu), imidazole and imidazole HCl salt, mayprovide improvements to the reaction profile, reaction rate and yield.

Compounds

The term “alkyl” refers to a monovalent straight or branched chain,saturated aliphatic hydrocarbon radical having a number of carbon atomsin the specified range. Thus, for example, “C₁₋₆ alkyl” refers to any ofthe hexyl alkyl and pentyl alkyl isomers as well as n-, iso-, sec- andtert-butyl, n- and iso-propyl, ethyl, and methyl. As another example,“C₁₋₄ alkyl” refers to n-, iso-, sec- and tert-butyl, n- and isopropyl,ethyl, and methyl.

The term “cycloalkyl” refers to any monocyclic ring of an alkane havinga number of carbon atoms in the specified range. Thus, for example,“C₃₋₈ cycloalkyl” (or “C₃-C₈ cycloalkyl”) refers to cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

The term “halogen” (or “halo”) refers to fluorine, chlorine, bromine andiodine (alternatively referred to as fluoro, chloro, bromo, and iodo orF, Cl, Br and I).

The term “haloalkyl” refers to an alkyl group as defined above in whichone or more of the hydrogen atoms have been replaced with a halogen(i.e., F, Cl, Br or I). Thus, for example, “C₁₋₆ haloalkyl” (or “C₁-C₆haloalkyl”) refers to a C₁ to C₆ linear or branched alkyl group asdefined above with one or more halogen substituents. The term“fluoroalkyl” has an analogous meaning except the halogen substituentsare restricted to fluoro. Suitable fluoroalkyls include the series(CH₂)₀₋₄CF₃ (i.e., trifluoromethyl, 2,2,2-trifluoroethyl,3,3,3-trifluoro-n-propyl, etc.).

The term “aryl” as a group or part of a group means phenyl or naphthyl.

The term “heteroaryl” as a group or part of a group means a 5- or6-membered aromatic ring having 1, 2 or 3 heteroatoms selected from N, Oand S, attached through a ring carbon or nitrogen. Examples of suchgroups include pyrrolyl, furanyl, thienyl, pyridyl, pyrazolyl,imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazinyl,pyrimidinyl, pyridazinyl, triazolyl, oxadiazolyl, thiadiazolyl,triazinyl and tetrazolyl.

As used herein, any alkyl group, cycloalkyl group, aryl group orheteroaryl group may be substituted, as indicated, by 0, 1, 2, 3, or 4substituents independently selected from the group consisting of C₁₋₆alkyl, C₁₋₆ alkenyl, C₁₋₆ alkynyl, aryl, halogen, —NH₂, and —OH.

The atoms in a compound described herein may exhibit their naturalisotopic abundances, or one or more of the atoms may be artificiallyenriched in a particular isotope having the same atomic number, but anatomic mass or mass number different from the atomic mass or mass numberpredominantly found in nature. The present invention is meant to includeall suitable isotopic variations of the compounds of described herein.For example, different isotopic forms of hydrogen (H) include protium (¹H) and deuterium (² H). Protium is the predominant hydrogen isotopefound in nature. Enriching for deuterium may afford certain therapeuticadvantages, such as increasing in vivo half-life or reducing dosagerequirements, or may provide a compound useful as a standard forcharacterization of biological samples.

Isotopically-enriched compounds described herein can be prepared withoutundue experimentation by conventional techniques well known to thoseskilled in the art or by processes analogous to those described in theSchemes and Examples provided herein using appropriateisotopically-enriched reagents and/or intermediates.

Compound Forms

Additional embodiments include compounds prepared by the methodsdisclosed herein. Particular embodiments include compounds selected fromthe group consisting of

A first compound embodiment is directed to

In an aspect of the first compound embodiment,

is substantially pure. Reference to “substantially pure” herein meansthe particular form makes up at least 50% of the compound present.

A second compound embodiment is directed to

is characterized by an X-ray powder diffraction pattern obtained usingcopper K_(α) radiation (i.e., the radiation source is a combination ofCu K_(α1) and K_(α2) radiation), which comprises three or morecharacteristic peaks. Characteristic peaks are illustrated in FIG. 1.

In a first aspect of the second compound embodiment,

is characterized by an X-ray powder diffraction pattern obtained usingcopper K_(α) radiation that comprises 2Θ values in degrees of about 2Θvalues (i.e., reflections at 2Θ values) in degrees of about 8.8, 11.7,and 20.1.

Reference to “about” with respect to 2Θ values provided herein indicates±0.1. In this embodiment and analogous embodiments that follow, the term“about” is understood to modify each of the 2Θ values; e.g., theexpression “about 8.8, 11.7, and 20.1” is short-hand for “about 8.8,about 11.7, and about 20.1”.

In a second aspect of the second compound embodiment,

is characterized by an X-ray powder diffraction pattern obtained usingcopper K_(α) radiation that comprises 2Θ values in degrees of about 2Θvalues (i.e., reflections at 2Θ values) in degrees of about 8.8, 11.7,14.8, 20.1, 23.7, and 27.7.

In a third aspect of the second compound embodiment,

is characterized by an X-ray powder diffraction pattern obtained usingcopper K_(α) radiation that comprises 2Θ values in degrees of about 2Θvalues (i.e., reflections at 2Θ values) in degrees of about 8.8, 11.7,14.8, 20.1, 23.7, 27.7, 29.0, 31.0 and 31.8.

In a fifth embodiment,

is substantially pure.

A third compound embodiment is directed to

is characterized by an X-ray powder diffraction pattern obtained usingcopper K_(α)radiation (i.e., the radiation source is a combination of CuK₆₀ ₁ and K_(α2) radiation), which comprises three or morecharacteristic peaks. Characteristic peaks are illustrated in FIG. 2.

In a first aspect of third compound embodiment,

is characterized by an X-ray powder diffraction pattern obtained usingcopper K_(α) radiation that comprises 2Θ values in degrees of about 2Θvalues (i.e., reflections at 2Θ values) in degrees of about 9.7, 13.7and 18.5.

In a second aspect of the third compound embodiment,

is characterized by an X-ray powder diffraction pattern obtained usingcopper K_(α) radiation that comprises 2Θ values in degrees of about 2Θvalues (i.e., reflections at 2Θ values) in degrees of about 9.2, 9.7,13.7, 15.0, 17.1, and 18.5.

In a third aspect of the third compound embodiment,

is characterized by an X-ray powder diffraction pattern obtained usingcopper K_(α) radiation that comprises 2Θ values in degrees of about 2Θvalues (i.e., reflections at 2Θ values) in degrees of about 9.2, 9.7,10.3, 13.7, 15.0, 17.1, 17.9, 18.5 and 20.9.

In a fourth aspect of the third compound embodiment,

is substantially pure.

A fourth compound embodiment is directed to

In a fifth aspect of the fourth compound embodiment,

is substantially pure.

A fifth compound embodiment is directed to Anhydrous Form I of

Anhydrous Form I is characterized by an X-ray powder diffraction patternobtained using copper K_(α) radiation (i.e., the radiation source is acombination of Cu K_(α1) and K_(α2) radiation), which comprises three ormore characteristic peaks. Characteristic peaks are illustrated in FIG.4.

In a first aspect of the fifth compound embodiment, Anhydrous Form I ischaracterized by an X-ray powder diffraction pattern obtained usingcopper K_(α) radiation that comprises 2Θ values in degrees of about 2Θvalues (i.e., reflections at 2Θ values) in degrees of about 8.7, 13.4,and 20.3.

In a second aspect of the fifth compound embodiment, Anhydrous Form I ischaracterized by an X-ray powder diffraction pattern obtained usingcopper K_(α) radiation that comprises 2Θ values in degrees of about 2Θvalues (i.e., reflections at 2Θ values) in degrees of about 5.0, 8.7,10.1, 13.4, 15.1, 17.6, and 20.3.

In a third aspect of the fifth compound embodiment, Anhydrous Form I ischaracterized by an X-ray powder diffraction pattern obtained usingcopper K_(α) radiation that comprises 2Θ values in degrees of about 2Θvalues (i.e., reflections at 2Θ values) in degrees of about 5.0, 8.7,10.1, 13.4, 15.1, 17.6, 20.3, 21.4, 22.2 and 23.3.

In a fourth aspect of the fifth compound embodiment, Anhydrous Form I issubstantially pure.

A sixth compound embodiment is directed to Anhydrous Form II of

Anhydrous Form II is characterized by an X-ray powder diffractionpattern obtained using copper K_(α) radiation (i.e., the radiationsource is a combination of Cu K_(α1) and K_(α2) radiation), whichcomprises three or more characteristic peaks. Characteristic peaks areillustrated in FIG. 6.

In a first aspect of the sixth compound embodiment, Anhydrous Form II ischaracterized by an X-ray powder diffraction pattern obtained usingcopper K_(α) radiation that comprises 2Θ values in degrees of about 2Θvalues (i.e., reflections at 2Θ values) in degrees of about 14.7, 18.9,and 22.8.

In a second aspect of the sixth compound embodiment, Anhydrous Form IIis characterized by an X-ray powder diffraction pattern obtained usingcopper K_(α) radiation that comprises 2Θ values in degrees of about 2Θvalues (i.e., reflections at 2Θ values) in degrees of about 7.2, 9.3,10.9, 11.3, 14.7, 18.9, 22.8, 23.8, and 25.1.

In a third aspect of the sixth compound embodiment, Anhydrous Form II ischaracterized by an X-ray powder diffraction pattern obtained usingcopper K_(α) radiation that comprises 2Θ values in degrees of about 2Θvalues (i.e., reflections at 2Θ values) in degrees of about 7.2, 9.3,11.3, 14.7, 18.9, and 22.8.

In a fourth aspect of the sixth compound embodiment, Anhydrous Form IIis substantially pure.

A seventh compound embodiment is directed to a crystalline isopropylalcohol solvate/hydrate (IPA solvate/hydrate) of

The IPA solvate/hydrate is characterized by an X-ray powder diffractionpattern obtained using copper K_(α) radiation (i.e., the radiationsource is a combination of Cu K_(α1) and K_(α2) radiation), whichcomprises three or more characteristic peaks. Characteristic peaks areillustrated in FIG. 9.

In a first aspect of the seventh compound embodiment, the IPAsolvate/hydrate is characterized by an X-ray powder diffraction patternobtained using copper K_(α) radiation that comprises 2Θ values indegrees of about 2Θ values (i.e., reflections at 2Θ values) in degreesof about 8.4, 16.0, and 23.7.

In a second aspect of the seventh compound embodiment, the IPAsolvate/hydrate is characterized by an X-ray powder diffraction patternobtained using copper K_(α) radiation that comprises 2Θ values indegrees of about 2Θ values (i.e., reflections at 2Θ values) in degreesof about 8.4, 16.0, 17.7, 19.1, 23.7, and 25.5.

In a third aspect of the seventh compound embodiment, the IPAsolvate/hydrate is characterized by an X-ray powder diffraction patternobtained using copper K_(α) radiation that comprises 2Θ values indegrees of about 2Θ values (i.e., reflections at 2Θ values) in degreesof about 6.7, 8.4, 13.4, 14.9, 15.6, 16.0, 17.7, 19.1, 19.8, 23.7, and25.5.

In a fourth aspect of the seventh compound embodiment, the IPAsolvate/hydrate is substantially pure.

EXAMPLES

The examples provided below are intended to illustrate the invention andits practice. Unless otherwise provided in the claims, the examples arenot to be construed as limitations on the scope or spirit of theinvention.

Abbreviations

-   -   ¹³C NMR Carbon-13 nuclear magnetic resonance spectroscopy    -   ¹H NMR Proton nuclear magnetic resonance spectroscopy    -   AcOH Acetic acid    -   aq., aq Aqueous    -   BOC tert-Butoxycarbonyl    -   Br₂ Bromine    -   Bu Butyl, C₄H₉    -   CDCl₃ Deuterated chloroform    -   CDI Carbonyl diimidazole    -   CH₂Cl₂ Dichloromethane    -   Cu—I, CuI Copper (I) iodide    -   DAP 1,3-Diaminopropane    -   DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene    -   DIPEA, DIE Diisopropyl ethyl amine (Hunig's base)    -   DMAc Dimethylacetamide    -   DMPU 1,3-Dimethyl-3,4,5,6-tetrahydro-2(1 H)-pyrimidinone    -   DMSO Dimethylsulfoxide    -   EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide    -   eq Equivalents, stoichiometric equivalents    -   Et Ethyl, C₂H₅    -   EtOAc Ethyl acetate    -   g Grams    -   h Hours    -   H₂ Hydrogen gas, hydrogen gas atmosphere    -   H₃PO₄ Phosphoric acid    -   HATU        1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium        3-oxide hexafluorophosphate    -   HCl Hydrochloric acid    -   HexLi Hexyl lithium    -   HPLC High performance liquid chromatography    -   IPA, i-PrOH Isopropyl alcohol    -   KF Karl Fischer titration    -   kg Kilogram    -   L Liter    -   LiNH₂ Lithium amide    -   M Molar    -   Me Methyl, CH₃    -   MeCN, CH₃CN Acetonitrile    -   MeMgBr Methylmagnesium bromide    -   MeMgCl Methylmagnesium chloride    -   MeOAc Methyl acetate    -   MeOH, CH₃OH Methanol, CH₃OH    -   MeSO₃H Methanesulfonic acid    -   MeTHF, 2-MeTHF 2-Methyl tetrahydrofuran    -   mg Milligram    -   MHz Megahertz    -   min Minutes    -   mL, ml milliliter    -   mM Millimolar    -   mm Millimeter    -   mmole millimole    -   Mol mole    -   MTBE Methyl tert-butyl ether    -   N Normal    -   N₂ Nitrogen gas, nitrogen gas atmosphere    -   Na₂CO₃ Sodium carbonate    -   Na₂S₂O₃ Sodium thiosulfate    -   NaCl Sodium chloride    -   NaOH Sodium hydroxide    -   NH₄Cl Ammonium chloride    -   nm Nanometer    -   nM Nanomolar    -   NMP N-Methyl-2-pyrrolidone    -   Pd/C Palladium on carbon    -   ppm Parts per million    -   psig Pounds per square inch [gauge],    -   1 Pascal=0.000145037738007 psig    -   pTSA p-Toluenesulfonic acid    -   Py Pyradine    -   RT, rt Room temperature or ambient temperature, approximately        25° C.    -   TBA tert-Butylamine    -   TEA Triethylamine    -   TFAA Trifluoroacetic anhydride    -   THF Tetrahydrofuran    -   UHP Urea hydrogen peroxide    -   V, v, vol Volume    -   v/v Volume per volume    -   w/w Weight per weight    -   wt % Percent by weight with respect to weight    -   x Refers to the number of times a process is iterated (e.g.,        “washed 3×”=“washed three times”)    -   μl Microliter    -   μm Micromillimeter, micron    -   μs Microsecond

Example 1 Epoxide Opening

A 1-L 3-neck round-bottom flask equipped with an overhead stirrer, acondenser, an additional funnel, and a N₂ inlet was charged with 2-MeTHF(290 mL) and magnesium turnings (9.0 g, 0.37 mol), followed by iodine(0.45 g, 0.002 mol). The mixture was heated to 70° C. and agitated for1.5 h. 4-Bromo-1-butene (47.5 g, 0.352 mol) was added dropwise over 1.5h at 70° C. The mixture was aged 6 h at 70° C., and then cooled to RT.

A 1-L 3-neck round-bottom flask equipped with an overhead stirrer, anadditional funnel, and a N₂ inlet was charged with(S)-(+)-epichlorohydrin (25.0 g, 0.27 mol), 2-MeTHF (150 mL) and Cu—I(2.56 g, 0.013 mol). The mixture was cooled to −60° C. The abovesolution of Grignard reagent was added via addition funnel over 1 h to 2h while maintaining the reaction temperature <−50° C. The reactionmixture was aged for additional 1 h at <−50° C. Then, the reactionmixture was transferred to a solution of 5.6 M aq. NH₄Cl (400 mL) viacannula. The quenched mixture, at 10° C., was warmed to RT and stirredfor additional 30 min. The layers were separated, and the organic phasewas washed with 3.5 M aq. NH₄Cl (150 mL) followed by 10% (w/w) NaClsolution (100 mL). The organic phase was azeotropically distilled undervacuum to a volume of 150 mL and flushed with MeTHF (3×100 mL). Thesolution was assayed for 36.9 g of the desired product, 91.7% assayyield. The dried solution of chlorohydrin in MeTHF solution was useddirectly in the next reaction.

¹H NMR (500 MHz, CDCl₃) δ 5.80 (m, 1 H), 5.03 (d, J=17.1 Hz, 1 H), 4.98(d, J =10.1 Hz, 1 H), 3.82 (m, 1 H), 3.64 (dd, J=11.1, 3.2 Hz, 1 H),3.48 (dd, J=11.1, 7.1 Hz, 1 H), 2.18 (s, br, 1 H), 2.10 (m, 2 H), 1.59(m, 1 H), 1.55 (m, 2 H), 1.48 (m, 1 H).

¹³C NMR (125 MHz, CDCl₃) δ 138.5, 115.2, 71.5, 50.7, 33.8, 33.7, 25.0.

Example 2 Preparation of Cyclopropyl Amide

A 2-L 3-neck round-bottom flask equipped with a condenser, an overheadstirrer under N₂ was charged with 2-methyl-THF (600 mL, KF<300) andsodium tert-butoxide (61.9 g, 0.644 mol). The mixture was stirred for 30min and then cooled to 0° C. to 5° C.

To a 2-MeTHF solution of chlorohydrin from Step 1 (36.9 g assay, 0.248mol, KF=220) from Example 1 was addeddiethyl[2-(dimethylamino)-2-oxoethyl] phosphonate (80.2 g, 86 wt %,0.309 mol). This mixed solution (˜230 mL) was transferred via cannula tothe above sodium tert-butoxide solution over 10 min with the temperaturerising to 18° C. The reaction solution was heated to 78° C. and aged for20 h. The reaction solution was cooled to RT (20° C.), and water (375mL) was added dropwise, while the internal temperature was maintained at<25° C. with external cooling. The separated organic phase was washedwith 10% w/w NaCl solution (3×100 mL). The organic phase was thenazeotropically distilled under vacuum at a pot temperature of 23° C. to28° C. to a volume of 200 mL. The organic phase was assayed (HPLC) for37.0 g of cyclopropyl amide, 82% assay yield. The dried solution ofcyclopropyl amide in MeTHF was directly used in the subsequent reactionwithout further purification. For epoxide intermediate:

¹H NMR (500 MHz, CDCl₃) δ 5.82 (m, 1 H), 5.04 (d, J=17.3 Hz, 1 H), 4.98(d, J =10.2 Hz, 1 H), 2.93 (m, 1 H), 2.76 (m, 1 H), 2.48 (m, 1 H), 2.13(m, 2 H), 1.57 (m, 4 H).

¹³C NMR (125 MHz, CDCl₃) δ 138.5, 115.0, 52.4, 47.3, 33.6, 32.1, 25.4.

For N,N-dimethyl amide product:

¹H NMR (500 MHz, d₄-MeOH) δ 5.81 (m, 1 H), 5.00 (m, 1 H), 4.93 (m, 1 H),3.20 (s, 3 H), 2.94 (s, 3 H), 2.09 (m, 2 H), 1.67 (m, 1 H), 1.52 (m, 2H), 1.37 (m, 1 H), 1.25 (m, 1 H), 1.07 (m, 1 H), 0.67 (m, 1 H).

¹³C NMR (125 MHz, d₄-MeOH) δ 175.8, 140.0, 115.2, 38.0, 36.4, 34.6,33.7, 29.9, 23.2, 19.7, 15.5.

Example 3 Methyl Grignard Addition

A 500-mL round-bottom flask under N₂ was charged with 3 M MeMgCl in THF(135 mL, 0.404 mol). The solution was heated to 60° C. The driedsolution of cyclopropyl amide in 2-MeTHF solution from Example 2 (36.6 gassay, 0.202 mol, ˜200 mL, KF<500) was added dropwise to the Grignardsolution over 2.5 h. After addition, the reaction solution was aged at60° C. for additional 1 h. The reaction was quenched to a solution of5.7 M aq. NH₄Cl (460 mL) and hexanes (425 mL), while the internaltemperature was maintained between 20° C. to 25° C. with externalcooling. The quenched mixture was stirred at RT for additional 1 h. Theseparated organic phase was washed with 1 N HCl (100 mL) followed by 10%w/w NaCl solution (100 mL). The organic phase was azeotropicallydistilled under vacuum at a volume of ˜60 mL, while maintaining theinternal temperature at <15° C. 27.5 g of assayed cyclopropyl methylketone, 90% assay yield.

¹H NMR (500 MHz, CDCl₃) δ 5.78 (m, 1 H), 5.00 (d, J=17.1 Hz, 1 H), 4.94(d, J =9.92 Hz, 1 H), 2.21 (s, 3 H), 2.06 (m, 2 H), 1.69 (m, 1 H), 1.48(m, 2 H), 1.38 (m, 1 H), 1.34 (m, 2 H), 1.32 (m, 1 H), 1.23 (m, 1 H).

¹³C NMR (125 MHz, CDCl₃) δ 208.4, 138.7, 114.8, 33.5, 32.8, 30.4, 29.4,28.6, 25.9, 18.2.

Example 4 Bromination, Method A

To a solution of methyl ketone in hexane from Example 3 (10.0 g assay,65.7 mmol, containing ˜1 vol hexanes, KF<200 ppm) was added CH₂Cl₂ (100mL). The solution was cooled to −45° C. to −50° C. Br₂ (10.50 g, neat,65.7 mmol) was added dropwise over 1 h via syringe pump, whilemaintaining the temperature between −45° C. to −50° C. After additional20 min aging, a second portion of Br₂ (3.15 g, 19.8 mmol) was addeddropwise between −45° C. to −50° C. over 20 min. The reaction mixturewas agitated for additional 20 min. DIPEA (2.12 g, 16.4 mmol) was addeddropwise over 15 min, maintaining the internal temperature between −45°C. to −50° C. The reaction mixture was then inverse-quenched to asolution of 10% Na₂S₂O₃ in 5% NaCl aq. (50 mL). The reaction mixture waspH adjusted to 4-6 with 1.0 N HCl (˜10 mL to 20 mL). After stirring for20 min, the organic phase (the bottom layer) was separated and washedwith water (30 mL). The organic phase was azeotropicallysolvent-switched to EtOAc (160 mL) below 15° C. until the KF of themixture <500 ppm. Typical assay yield is 85% to 89%. The dried solutionof bromo ketone in EtOAc was directly used in the next reaction withoutfurther purification.

The bromide product is a mixture of diastereomers.

¹H NMR (500 MHz, CDCl₃) δ 4.16 (m, 1 H), 3.85 (m, 1 H), 3.62 (m, 1 H),2.23 and 2.20 (s, 3 H), 2.18 (m, 1 H), 1.81 (m, 1 H), 1.73 (m, 2 H),1.53 (m, 1 H), 1.38 (m, 3 H), 1.26 (m, 1 H), 0.77 (m, 1 H).

¹³C NMR (125 MHz, CDCl₃) δ 208.17, 208.15, 52.82, 52.79, 36.30, 36.27,35.71, 35.67, 32.50, 32.47, 30.50, 30.45, 29.24, 29.12, 26.61, 26.53,25.48, 25.41, 18.16, 18.05.

Example 5 Bromination, Method B

To a solution of methyl ketone in hexane from Example 3 (10 g assay,65.7 mmol, ˜50 wt %) was added MeCN (30 mL) followed by pyridine (2.6 g,32.85 mmol). The batch was cooled to −5° C. to 5° C. A solution ofpyridinium tribromide (25.2 g, 92 mmol) in MeCN (50 mL) was addeddropwise over several hours, while maintaining the batch temperaturebetween −5° C. and 5° C. After aging the reaction slurry for additional30 min, a solution of 10% Na₂S₂O₃ in 5% NaCl aq. (30 mL) was slowlycharged. The batch was agitated at RT for at least 30 min. EtOAc (60 mL)was charged to the separated organic layer, followed by 0.5N HCl (40mL). The organic phase was then washed with 15% aq. NaCl (20 mL). Theorganic phase was aezotropically dried and solvent-switched to EtOAc ata final volume of ˜80 mL under vacuum. Typical assay yield is 91 to 93%.The crude stream of bromo ketone may be used directly in the subsequentstep without further purification.

Example 6 Baeyer-Villiger Oxidation

The solution of di-Br ketone (20.0 g assay, 64.1 mmol) in EtOAc (160 mL,KF<500 ppm) from Example 4 was cooled to 0° C. UHP (24.1 g, 256.4 mmol)was added in portions. TFAA (55.2 g, 262.8 mmol) was then added dropwiseunder N₂ over 3 h, while the internal temperature was maintained between0° C. to 3° C. The resulting mixture stirred overnight at 0° C. (˜16 h)until HPLC showed that conversion was greater than >95%. The reactionmixture was pH adjusted to 7-8 by addition of 20% Na₂CO₃ (˜140 mL, 265mmol), while the internal temperature was maintained below 5° C. withexternal cooling. The organic phase was separated. The aqueous layer wasextracted with EtOAc (100 mL). The combined organic phase was washedwith 10% Na₂S₂O₃ in 5% NaCl aq. (w/w, 60 mL) followed by 10% NaCl aq.solution (60 mL). The organic layer was azeotropically solvent-switchedto hexanes (100 mL) until the content of EtOAc is <1% (by ¹H NMR assay)and KF<500 ppm. The slurry was agitated at 20° C. for additional 1 h;the solid was removed through filtration. The wet cake was washed withhexanes (3×20 mL). The combined filtrate was concentrated to a volume of˜50 mL to 60 mL (contained ˜1 vol of hexane). Typical assay yield is92%-95% (the combined yield). The concentrated crude process stream wasused directly for the next reaction. The bromide acetate product is amixture of diastereomers.

¹H NMR (500 MHz, CDCl₃) δ 4.18 (m, 1 H), 3.87 (m, 1 H), 3.84 (m, 1 H),3.65 (m, 1 H), 2.19 (m, 1 H), 2.03 (s, 3 H), 1.83 (m, 1 H), 1.73 (m, 1H), 1.58 (m, 1 H), 1.36 (m, 1 H), 1.29 (m, 1 H), 1.04 (m, 1 H), 0.86 (m,1 H), 0.57 (m, 1 H).

¹³C NMR (125 MHz, CDCl₃) δ 171.9, 54.53, 54.47, 53.0, 36.49, 36.46,35.87, 35.82, 30.8, 30.7, 26.20, 26.12, 21.14, 18.40, 18.37, 12.21,12.12.

The bromide alcohol product is a mixture of diastereomers.

¹H NMR (500 MHz, CDCl₃) δ 4.39 (m, 1 H), 3.89 (m, 1 H), 3.86 (m, 1 H),3.65 (m, 1 H), 3.24 (m, 1 H), 2.19 (m, 1 H), 1.84 (m, 2 H), 1.69 (m, 1H), 1.54 (m, 1 H), 1.30 (m, 1 H), 1.18 (m, 1 H), 0.95 (m, 1 H), 0.74 (m,1 H), 0.36 (m, 1 H).

¹³C NMR (125 MHz, CDCl₃) δ 53.11, 53.06, 52.9, 52.8, 36.5, 35.9, 35.8,31.1, 31.0, 26.44, 26.38, 20.81, 20.78, 14.8, 14.7.

Example 7 De-Bromination, Method A

A 1-L 3-neck round-bottom flask equipped with an overhead stirrer, anadditional funnel, and a N₂ inlet was charged with DAP (151 mL). Thesolution was cooled to 0° C. HexLi (2.3 M, 188 mL, 433 mmol) was chargeddropwise over 1 h, maintaining the temperature below 5° C. After theaddition, the reaction mixture was stirred at 0° C. for additional 30min. A solution of bis-Br esters (total 72.2 mmol assay, ˜50 mL of ˜50%v/v solution in hexane) prepared from Example 6 was added to the aboveslurry mixture dropwise over 1 h, while maintaining internal temperatureat 0° C.±2° C. The reaction mixture was stirred at 0° C. for additional0.5 h to 1 h.

A 1-L 3-neck round-bottom flask equipped with an overhead stirrer wascharged with 10% aq. NH₄Cl (240 mL) and MTBE (240 mL). The solution wascooled to 0° C. Then, the debrominated reaction mixture was transferredto the cold solution of NH₄Cl/MTBE through flexible tubing under partialvacuum with a good mixing over 20 min, while the internal temperaturewas maintained below 5° C. The debromination reaction flask and tubingwere rinsed/washed with a cold solution of NH₄Cl (10%, 24 mL)/MTBE (24mL). The resulting quenched mixture was stirred at 5° C. for additional30 min until the solid was dissolved. The organic phase was retained,and the aqueous layer was further extracted with MTBE (120 mL). Thecombined organic phase was washed with 10% NH₄Cl aq. (120 mL), followedby 10% NaCl (120 mL). The organic phase was concentrated andsolvent-switched into MeOH to a volume of ˜60 mL at <15° C. underreduced pressure. The reaction solution was cooled to 0° C. NaOH aq.solution (1 N, 50 mL, 50 mmol) was added dropwise, maintaining theinternal temperature at 0° C. to 5° C. The reaction mixture was stirredat 0° C. for additional 0.5 h to 1 h. 10% NH₄Cl aq. solution (120 mL)and MTBE (120 mL) were added. The two phases were separated. Theseparated aqueous phase was back-extracted with MTBE (120 mL). Thecombined organic phase was washed with 10% NaCl (40 mL). The organicphase was azeotropically solvent-switched to EtOAc to a final volume of˜30 mL until KF<500 ppm. Typical assay yield is 80% to 84%. The crudeprocess stream was used directly for the next reaction.

¹H NMR (500 MHz, CDCl₃) δ 3.20 (m, 1 H), 2.31 (s, br, 1 H), 2.21 (m, 2H), 1.92 (t, J=2.6 Hz, 1 H), 1.60 (m, 2 H), 1.33 (m, 1 H), 1.21 (m, 1H), 0.89 (m, 1 H), 0.68 (m, 1 H), 0.32 (m, 1 H).

¹³C NMR (125 MHz, CDCl₃) δ 84.7, 68.6, 52.7, 30.7, 28.0, 20.4, 18.2,14.5.

Example 8 De-Bromination, Method B

A 250-mL 3-neck round-bottom flask equipped with an overhead stirrer anda N₂ inlet was charged with DAP (120 mL). Then, LiNH₂ (6.83 g), 297mmol) was charged in 2 portions (slightly exothermic). The resultingsuspension was stirred at 22° C. to 25° C. for 1 h to 2 h, and then thesuspension was cooled to 0° C. A solution of bis-Br esters (total 45.7mmol assay, ˜40 ml, of ˜50% v/v solution in THF, KF<500 ppm) preparedfrom the Example 6 was added to the above slurry mixture dropwise over 1h, while maintaining batch internal temperature at 0° C.±3° C. Thereaction mixture was stirred at 0° C. for additional 2 h to 3 h. Thereaction mixture was warmed to 22° C. to 25° C. over 2 h to 3 h and agedat 22° C. to 25° C. for additional 5 h to 6 h until molar conversion wasgreater than 95%. Then, the reaction mixture was cooled to 0° C. andquenched to a cold solution (0° C.) of 15% aq. NH₄Cl (150 mL) and MTBE(150 mL) through flexible tubing under partial vacuum with a good mixingover 0.5 h, while the internal temperature was maintained below 5° C.The debromination reaction flask and tubing were rinsed/washed with thecold solution of NH₄Cl (15%, 24 mL)/MTBE (24 mL). Then, the resultingquenched mixture was stirred at 20° C. for additional 30 min until theentire solid was dissolved. The organic phase was retained, and theaqueous layer was further extracted with MTBE (75 mL). The combinedorganic phase was washed with 15% NH₄Cl aq. (75 mL), followed by 10%NaCl (75 mL). The organic phase was azeotropically solvent-switched toEtOAc to a final volume of ˜30 mL until KF<500 ppm. Typical assay yieldis 72% over 2 steps (Baeyer-Villiger oxidation & debromination). Thecrude process stream may be used directly for the next reaction.

Example 9 De-Bromination, Method C

n-BuLi (2.5 M in hexanes, 25.8 mL, 64.61 mmol) was added dropwise to asolution of 1,3-diaminopropane (2.52 g, 33.97 mmol) in 2-MeTHF (36 mL)at −25° C. to −15° C. under N₂, while maintaining the batch temperaturebelow −15° C. The resulting slurry was agitated at −15° C.±5° C. for 1h, warmed to between 20° C. to 25° C. over 2 h and agitated foradditional 1 h. The batch was then cooled to between 0° C. to 25° C. Asolution of bis-bromides prepared from the Example 6 (9.23 mmol assay ofcombined) was added dropwise over 1-5 h, while maintaining the batchtemperature around 0° C. to 25° C. DMPU (4.73 g, 36.92 mmol) was added,and the batch was agitated at 20° C. to 25° C. for several hours. Water(40 mL) was added slowly, while maintaining the internal temperaturebetween 15° C. to 30° C. The batch was agitated at RT for additional 2 hto 4 h, and filtered through SOLKA-FLOC® (powdered cellulose,International Fiber Corporation, North Tonawanda, N.Y.). The wet cakewas washed with 2-MeTHF/hexanes (1:1 v/v, 9 mL). The organic phase wasseparated, and the aqueous layer was extracted with 2-MeTHF/hexanes (1:1v/v, 18 mL). The combined organic phase was washed with 20% NH₄Cl (15mL) followed by water (2×15 mL). The organic phase was azeotropicallydried and solvent-switched to 2-MeTHF at a final volume of ˜5 mL.Typical yield ˜95%. The concentrated crude process stream may be useddirectly for the subsequent step without further purification.

Example 10 Carbamate Formation, Method A

A slurry of CDI (7.6 g, 44.3 mmol) in EtOAc (75 mL) was cooled to 0° C.A solution of alkyne alcohol (5.0 g active assay, 40.3 mmol) in EtOAc(7.5 mL) from Example 7 was added slowly in about 1 h, while maintainingthe temperature at 0° C. The reaction mixture was stirred at 0° C. foradditional 1 h. Water (40 mL) was added slowly at 0° C., and resultingmixture was stirred at 0° C. for 1 h. The organic phase was separated.The organic phase was azeotropically dried to a KF<250 ppm with EtOAc invacuum at below 20° C. (internal temperature). The solvent was thenazeotropically switched to NMP at a final volume of ˜95 mL.L-tert-leucine (6.3 g, 48.4 mmol) and 2-hydroxypyridine N-oxide (1.79 g,16.1 mmol) were added to the above anhydrous solution. The reactionmixture was heated up to between 60° C. to 65° C. and stirred for 10 hto 15 h. The reaction mixture was cooled to 0° C., and water (75 mL) wasslowly added. 5 N HCl solution (30 mL) was added slowly to adjust pH to˜2 by pH meter. The two phases were separated. The aqueous phase wasback-extracted with MTBE (75 mL) twice. The organic phase was washedwith water (75 mL) and then azeotropically distillated at a final volumeof ˜95 mL. IPA (16 mL, 3.2 v) was added, and the solution was heated to40° C. At 40° C., TBA (1.85 mL, 17.5 mmol) was slowly added over about30 min. The slurry was stirred at 40° C. for about 30 min to form a seedbed. Additional TBA (3.69 mL, 34.9 mmol) was slowly added at 40° C. inabout 1 h. After aging at 40° C. for additional 1 h, the slurry wasslowly cooled to 20° C. and aged for 1 h. The slurry was further cooledto 0° C. and stirred for 1 h before filtration. The wet cake washed with5% IPA/MTBE solution (2×30 mL, pre-cooled to 0° C.). The wet cake wasdried under vacuum at 40° C. with N₂ sweep to give 12.4 g of TBA salt.

¹H NMR (400 MHz, CDCl₃) δ 8.19 (s, br, 3 H), 6.04 (d, J=8.4 Hz, 1 H),3.60 (m, 1 H), 3.45 (d, J=8.4 Hz, 1 H), 2.72 (t, J=2.7 Hz, 1 H), 2.18(td, J=7.1, 2.7 Hz, 2 H), 1.54 (m, 2 H), 1.26 (m, 2 H), 1.22 (s, 9 H),0.89 (m, 1 H), 0.88 (s, 9 H), 0.76 (m, 1 H), 0.44 (m, 1 H).

¹³C NMR (100 MHz, CDCl₃) δ 172.9, 156.1, 84.4, 71.2, 64.2, 53.5, 50.0,34.0, 29.7, 27.7, 27.18, 27.15, 17.6, 17.3, 11.3.

An X-ray powder diffraction pattern was generated to characterize themolecular structure of the TBA salt of(S)-3,3-dimethyl-2-(((1R,2R)-2-(pent-4-ynyl)cyclopropoxy) carbonylamino)butanoic acid. The pattern was generated on a Philips Analytical X'PertPRO X-ray Diffraction System with PW3040/60 console, using a PW3373/00ceramic Cu LEF X-ray tube K-Alpha radiation as the source. The pattern,shown in FIG. 1, exhibited characteristic reflections corresponding tod-spacings as follows.

Position d-spacing [° 2 θ] [{acute over (Å)} ({acute over (Å)} = 0.1nm)] 8.8 10.1 20.1 4.4 11.7 7.6 27.7 3.2 14.8 6.0 23.7 3.8 29.0 3.1 31.82.8 31.0 2.9

Example 11 Carbamate Formation, Method B

A solution of alkyne alcohol (3.2 g assay, 25.85 mmol) in 2-MeTHF fromExample 7 was added to a mixture of CDI (5.62 g, 33.61 mmol) in 2-MeTHF(32 mL) at 0° C.±5° C. over 2 h to 5 h. The reaction solution wasagitated for additional 1 h to 2 h. Water (16 mL) was added dropwise,while maintaining the internal temperature at 0° C. to 5° C. Thereaction solution was then agitated at 0° C. to 5° C. for additional 1 hto 2 h. Heptane (38 mL) was added. The organic phase was separated, andthe aqueous layer was extracted with 2-MeTHF/heptane (1:1, 10 mL). Thecombined organic phase was washed with water (16 mL). The organic phasewas azeotropically dried and solvent-switched to 2-MeTHF at a finalvolume of ˜10 mL. NMP (56 mL) was added, followed by tert-L-leucine(4.19 g, 31.02 mmol) and 2-hydroxypyridine N-oxide (1.17 g, 10.34 mmol).The reaction mixture was agitated between 60° C. to 65° C. for 10 h to18 h. MTBE (48 mL) and water (48 mL) were added at RT. The batch was pHadjusted with 5N HCl to pH=2.0-2.5. The organic phase was separated, andthe aqueous layer was extracted with MTBE (48 mL). The combined organicphase was washed with water (2×32 mL). The organic phase was extractedwith NaOH aq. (1 N, 40 mL). The separated aqueous phase was washed withMTBE (2×32 mL). MTBE (50 mL) was added, and the batch was pH adjusted topH=2.0-2.5. The organic phase was washed with water (16 mL). The organicphase was azeotropically dried under reduced pressure at a final volumeof ˜110 mL containing ˜1 wt % water. A solution of TBA (2.48 g, 33.61mmol) in MTBE (3 mL) was added dropwise at 40° C.±5° C. After ˜35% ofthe above TBA solution was added, the batch was seeded. The remainingTBA solution was added dropwise over 2 h to 4 h. After aging at 40°C.±5° C. for additional 1 h to 2 h, the batch was cooled to RT andfiltered. The wet cake was washed MTBE (3×30 mL) and dried in a vacuumoven at 30° C. to 35° C. with N₂ sweep, which afforded the TBA salt.Typical yield: 76-81%.

Example 12 Preparation of(2S,4R)-4-(3-chloro-7-methoxyquinoxalin-2-yloxv)-2-(methoxycarbonyl)pyrrolidiniummethanesulfonate

To a slurry of 2,3-dichloroquinoxaline (100 g, 0.437 mol) andN-Boc-4-trans-hydroxy-L-proline methyl ester (118 g, 0.48 mol) in DMAc(500 ml, KF<150) at RT, DBU (86 g, 0.568 mol) was added. The slurry wasagitated at 40° C. to 45° C. for ˜35 h. The batch was then cooled to 15°C. EtOAc (1.2 L) followed by citric acid (10%, 504 mL, 162 mmol) wasadded while the internal temperature was maintained at <25° C. Theorganic phase was washed with a solution of 10% citric acid (200 mL) andwater (200 mL) followed by water (2×400 mL). The organic phase wasazeotropically dried and solvent-switched to MeCN at a final volume of˜880 mL. MeSO₃H (36 mL, 0.555 mol) was added, and the reaction mixturewas aged at 40° C. for ˜16 h. To the reaction slurry, MTBE (1.05 L) wasadded dropwise over 2 h at 35° C. Then, the batch was further cooled to0° C. to 5° C. and aged for 2 h to 3 h before filtration. The wet cakewas displacement washed with 30% MeCN in MTBE (2×600 mL) and vacuum-ovendried at 40° C. to give the product.

¹H NMR (400 MHz, d₆-DMSO) δ 9.74 (s, br, 2 H), 7.86 (d, J=9.2 Hz, 1 H),7.34 (dd, J=9.2, 2.8 Hz, 1 H), 7.26 (d, J=2.8 Hz, 1 H), 5.77 (m 1 H),4.69 (dd, J=10.6, 7.6 Hz, 1 H), 3.92 (s, 3 H), 3.89 (dd, J=13.2, 5.2 Hz,1 H), 3.81 (s, 3 H), 3.63 (m, 1 H), 2.71 (m, 1 H), 2.60 (m, 1 H), 2.35(s, 3 H).

¹³C NMR (100 MHz, d₆-DMSO) δ 168.3, 161.0, 151.8, 140.4, 135.4, 133.3,128.6, 119.8, 106.0, 75.6, 58.0, 56.0, 53.2, 50.5, 39.6, 33.9.

HPLC conditions: Hypersil Gold PFP column, 150×4.6 mm, 3.0 μm; Columntemperature of 40° C.; Flow rate of 1.8 mL/min; and Wavelength of 215nm.

Gradiant: min CH₃CN 0.1% H₃PO₄ 0 25 75 12 70 30 12.1 25 75 14 25 75Retention times: min. Dichloroquinoxaline 7.8 Proline quinoxaline 9.8De-BOC quinoxaline 3.6

Example 13 Preparation of(2S,4R)-4-(3-chloro-7-methoxyquinoxalin-2-yloxy)-2-(methoxycarbonyl)pyrrolidiniummethanesulfonate

To a slurry of 2,3-dichloroquinoxaline (20 g, 0.087 mol) andN-Boc-4-trans-hydroxy-L-proline methyl ester (23.6 g, 0.096 mol) in DMAc(100 ml) at RT, DBU (17 mL, 0.114 mol) was added. The slurry wasagitated at 50° C. for ˜40 h. The batch was then cooled to 15° C. EtOAc(240 mL) followed by citric acid (10%, 97 mL) was added while theinternal temperature was maintained at <25° C. The organic phase waswashed with a solution of 10% citric acid (40 mL) and water (40 mL)followed by water (2×80 mL). The organic phase was azeotropically driedand solvent-switched to MeCN at a final volume of ˜175 mL. MeSO₃H (7.2mL, 0.111 mol) was added, and the reaction mixture was aged at 56° C. to60° C. for 1 h to 3 h. The batch was seeded with 100 mg ofmethanesulfonate salt product seeds and aged for additional 7 h. To thereaction slurry, MTBE (120 mL) was added dropwise over 3 h at 55° C.Then, the batch was aged for 2 h, cooled to 0° C. to 5° C., and aged for2 h before filtration. The wet cake was displacement washed with 30%MeCN in MTBE (3×120 mL) and vacuum oven-dried at 40° C. with N₂ sweep togive the product.

An X-ray powder diffraction pattern was generated to characterize themolecular structure of the methanesulfonate salt of(2S,4R)-4-(3-chloro-7-methoxyquinoxalin-2-yloxy)-2-(methoxycarbonyl)pyrrolidine.Powder X-ray Diffraction data were acquired on a Panalytical X-pert ProPW3040 System configured in the Bragg-Brentano configuration andequipped with a Cu radiation source with monochromatization to Kαachieved using a Nickel filter. A fixed slit optical configuration wasemployed for data acquisition. Data were acquired between 2° and 40° 2θ.Samples were prepared by gently pressing powdered sample onto a shallowcavity zero background silicon holder. The pattern, shown in FIG. 2,exhibited characteristic reflections corresponding to d-spacings asfollows.

Position d-spacing [° 2 θ] [{acute over (Å)} ({acute over (Å)} = 0.1nm)] 9.2 9.6 9.7 9.1 10.3 8.6 13.7 6.5 15.0 5.9 17.1 5.2 17.9 5.0 18.54.8 20.9 4.2

Example 14 Preparation of(S)-2-(((1R,2R)-2-(5-(6-methoxy-3-((3R,5S)-5-(methoxycarbonyl)pyrrolidin-3-yloxy)quinoxalin-2-yl)pent-4-ynyl)cyclopropoxy)carbonylamino)-3,3-dimethylbutanoicacid and alkene macrocyclic ester

To a 3-neck flask were added CuI (0.219 g, 1.152 mmol),chloroquinoxaline MsOH salt from Example 12 (50 g, 115 mmol), alkyneacid TBA salt from Example 10 (49.3 g, 121 mmol), andbis(triphenylphosphine)palladium(II) dichloride (0.404 g, 0.573 mmol).The flask was vacuumed degassed with N₂. MeOH (500 ml) was added, andthe reaction mixture was vacuum degassed again with N₂. TEA (32.1 ml,230 mmol) was added. The reaction solution was aged at 35° C. for 3 h to5 h. The batch was then concentrated to a volume of ˜100 mL in vacuum.THF (250 mL) and EtOAc (250 mL) were added. The reaction mixture wascooled to below 5° C. HCl solution (1 N, ˜180 mL) was added slowly atbelow 5° C. until the reaction solution was pH adjusted to ˜2. NaCl aq.solution (10%, 350 mL) was added. The separated aqueous phase wasback-extracted with a solution of THF (250 mL) and EtOAc (250 mL). Thecombined organic phase was washed with 10% NaCl aq. solution (500 mL).The organic phase was azeotropically concentrated in vacuum with THF atbelow 20° C. until the KF of the solution was less than 500 ppm. Then,the reaction solvent was switched to DMAc (650 mL) in vacuum at below20° C.

A solution of HATU (55.1 g, 145 mmol) in DMAc (650 mL) at RT wasvacuumed degassed with N₂. The solution was then cooled to 0° C., andDIPEA (58.5 mL, 335 mmol) was added dropwise at below 0° C. to 5° C.Then, the above solution of alkyne quinoxaline acid (65 g assay, 112mmol) in DMAc was added dropwise over 10 h, while maintaining theinternal temperature at 0° C. After addition, the batch was agitated at0° C. for additional 2 h. EtOAc (750 mL) was added at below 5° C. Asolution of 10% NaCl aq. solution (400 mL), water (125 mL) and 1 N HClsolution (100 mL) was slowly added while maintaining the batchtemperature at below 5° C. The solution was then adjusted to pH=2 with 1N HCl (˜25 mL). The separated aqueous phase was back-extracted withEtOAc (500 mL). The combined organic phase was washed with 10% NaCl aq.solution (500 mL). After 10% NaCl aq. solution (500 mL) was added to thecombined organic phase, the mixed solution was cooled to 0° C. to 5° C.1 N NaOH aq. solution (˜25 mL) was added to adjust the pH=˜7. Theseparated organic phase was filtered through CELITE® (filter aid, FisherScientific, Fair Lawn, N.J.) and solvent-switched to IPA at a finalvolume of 300 mL. AcOH (5.0 mL) was added, and the batch was then heatedto reflux for 30 min. The slurry was cooled to 60° C., and water (250mL) was added dropwise over 1 h. After addition, the batch was aged foradditional 30 min before slowly cooling to RT in about 2 h. After agingat least 1 h, the batch was filtered. The wet cake was displacementwashed with 50% aq. IPA (100 mL). Suction-drying at RT afforded 56 g ofmacrocyclic alkyne ester.

¹H NMR (400 MHz, CDCl₃) δ 7.80 (d, J=9.2 Hz, 1 H), 7.17 (dd, J=9.2, 2.8Hz, 1 H), 7.04 (d, J=2.8 Hz, 1 H), 5.82 (t, J=4.2 Hz, 1 H), 5.26 (d,J=9.9 Hz, 1 H), 4.62 (dd, J=10.3, 7.3 Hz, 1 H), 4.51 (d, J=11.6 Hz, 1H), 4.40 (d, J=9.9 Hz, 1 H), 4.03 (dd, J=11.6, 4.4 Hz, 1 H), 3.91 (s, 3H), 3.87 (m, 1 H), 3.73 (s, 3 H), 2.85 (dt, J=12.1, 4.2 Hz, 1 H), 2.76(d, J=14.4, 7.3 Hz, 1 H), 2.49 (dt, J=12.2, 5.4 Hz, 1 H), 2.30 (ddd,J=14.6, 10.1, 4.2 Hz, 1 H), 1.99 (m, 1 H), 1.82 (m, 1 H), 1.74 (m, 1 H),1.08 (s, 9 H), 0.92 (m, 2 H), 0.76 (m, 1 H), 0.47 (m, 1 H).

¹³C NMR (100 MHz, CDCl₃) δ 172.3, 171.3, 161.2, 157.4, 156.3, 140.4,134.3, 130.2, 129.5, 119.5, 105.7, 98.9, 75.5, 75.2, 59.4, 58.1, 55.7,55.6, 54.1, 52.3, 35.3, 35.0, 29.9, 28.0, 26.3, 18.7, 18.3, 10.3.

IPC HPLC conditions: Ascentis Express C18 column, 100×4.6 mm, 2.7 μm;Column temperature of 40° C.; Flow rate of 1.8 mL/min; and Wavelength of215 nm.

Gradiant: min CH₃CN 0.1% H₃PO₄ 0 10 90 6 95 5 9 95 5 9.1 10 90 Retentiontimes: min. De-BOC quinoxaline 2.3 Alkyne quinoxaline acid 3.3 Alkynemacrocyclic ester 5.7

Example 14A Isolation of the Macrocvclic Alkene Ester Anhydrous Form Ifrom Anhydrous Form II and/or Solvate Form

A slurry of macrocyclic alkyne ester powder (2.9 g) from the prior stepin IPA (25 mL) was stirred at 20° C. for 2 h. The batch was filtered andwashed with IPA (6 mL). The wet cake was dried at 60° C. under vacuum togive macrocyclic alkyne ester anhydrous Form I, 96% yield.

An X-ray powder diffraction pattern was generated to characterize themolecular structure of the macrocyclic alkyne ester anhydrous Form I.The pattern was generated as described above in Example 10. The pattern,shown in FIG. 3, exhibited characteristic reflections corresponding tod-spacings as follows.

Relative Position d-spacing Intensity [° 2 θ] [{acute over (Å)} ({acuteover (Å)} = 0.1 nm)] [%] 5.0 17.5 19.9 8.7 10.1 76.1 10.1 8.8 29.2 13.46.6 100.0 15.1 5.9 23.7 17.6 5.1 44.0 20.3 4.4 64.2 21.4 4.1 11.6 22.24.0 8.8 23.3 3.8 22.3

In addition to the X-ray powder diffraction patterns described above,Anhydrous Form I was further characterized by solid-state carbon-13nuclear magnetic resonance (¹³C NMR) spectra. The ¹³C NMR spectra wererecorded using a Bruker 4 mm HXY triple resonance CPMAS, and a Bruker 4mm H/FX double resonance CPMAS probe, respectively. The ¹³C NMR spectrawere collected utilizing proton/¹³C variable-amplitudecross-polarization (VACP) with a contact time of 3 ms, and a pulse delayof 4 s, while magic-angle spinning (MAS) the samples at 13 kHz. A linebroadening of 30 Hz was applied to the ¹³C NMR spectra before FourierTransformation. Chemical shifts are reported on the TMS scale using thecarbonyl carbon of glycine (176.7 ppm.) as a secondary reference. FIG. 4shows the solid state ¹³C CPMAS NMR spectrum for the crystallineAnhydrous Form I. Characteristic peaks for Anhydrate I are observed at172.4, 171.7, 161.5, 157.7, 142.0, 135.3, 130.2, 112.7, 112.1, 97.3,77.2, 75.0, 60.3, 59.3, 57.0, 55.3, 54.4, 37.2, 34.9, 30.5, 27.9, 20.9,19.4, and 10.5 ppm.

Further, a differential scanning calorimetry (DSC) curve was prepared.DSC data are acquired using TA Instruments DSC 2910 or equivalent.Between 2 mg and 6 mg sample is weighed into a pan and covered. This panis then covered and placed at the sample position in the calorimetercell. An empty pan is placed at the reference position. The calorimetercell is closed and a flow of nitrogen is passed through the cell. Theheating program is set to heat the sample at a heating rate of 10°C./min to a temperature of approximately 300° C. The heating program isstarted. When the run is completed, the data are analyzed using the DSCanalysis program contained in the system software. The thermal eventsare integrated between baseline temperature points that are above andbelow the temperature range over which the thermal event is observed.The data reported are the onset temperature, peak temperature andenthalpy. FIG. 5 shows a typical DSC curve for the crystalline AnhydrousForm I.

Example 14B Preparation of Macrocyclic Alkyne Ester IsopropylAlcohol/Water Solvate and Anhydrous Form II from Anhydrous Form I

To a solution of IPA (62.1 mL), EtOAc (16.2 mL), and water (45 mL) wascharged macrocyclic alkyne ester anhydrous Form I powder (10 g). Theresulting slurry was stirred at 20° C. Then, macrocyclic alkyne estersolvate seeds (0.156 g) were added. The batch was aged at 50° C. to 65°C. for 30 min, and cooled to 10° C. over 2 h. After aging at 10° C. for1.5 h, the batch was heated to 50° C. to 65° C. and aged at 50° C. to65° C. for 3 h before cooling to 10° C. The heating-cooling cycle wasrepeated several times until the anhydrous Form I was converted to theIPA/water solvate. The solvate was filtered and washed with 40 mL of 50%aqueous IPA. The wet cake was dried at 60° C. under vacuum to give theanhydrous Form II. 95% yield. Anhydrous Form II can also be prepared byheating the corresponding IPA solvate/hydrate to >150° C.

An X-ray powder diffraction pattern was generated to characterize themolecular structure of the crystalline macrocyclic alkyne esteranhydrous Form II. The pattern was generated as described above and inExample 10. The pattern, shown in FIG. 6, exhibited characteristicreflections corresponding to d-spacings as follows.

Relative Position d-spacing Intensity [° 2 θ] [{acute over (Å)} ({acuteover (Å)} = 0.1 nm)] [%] 7.2 12.3 58.6 9.3 9.5 54.5 10.9 8.1 20.8 11.37.8 39.1 14.7 6.0 88.1 18.9 4.7 71.8 22.8 3.9 100.0 23.8 3.7 35.6 25.13.6 21.0

In addition to the X-ray powder diffraction patterns described above,the crystalline macrocyclic alkyne ester anhydrous Form II were furthercharacterized by solid-state carbon-13 nuclear magnetic resonance (¹³CNMR) spectra. The ¹³C NMR spectra were recorded as described above. FIG.7 shows the solid state ¹³C CPMAS NMR spectrum for the crystallinemacrocyclic alkyne ester anhydrous Form II. Characteristic peaks areobserved at 174.2, 171.7, 161.9, 158.6, 157.0, 140.7, 134.4, 131.4,129.6, 113.4, 111.3, 97.9, 75.6, 74.1, 61.3, 59.7, 57.2, 55.2, 53.2,37.8, 35.5, 32.6, 30.5, 28.3, 26.5, 19.7, 18.7 and 12.0 ppm.

Further, a differential scanning calorimetry (DSC) curve was prepared asdescribed above. FIG. 8 shows a typical DSC curve for the crystallinemacrocyclic alkyne ester anhydrous Form II.

Example 14C Preparation of Macrocyclic Alkyne Ester IsopropylAlcohol/Water Solvate

The IPA/water co-solvate/hydrate of macrocyclic alkyne ester is preparedin mixtures of IPA/water ranging from 4 wt % water in IPA at 15° C. toabove 12 wt % water in IPA at 60° C.

An X-ray powder diffraction pattern was generated to characterize themolecular structure of the crystalline IPA solvate/hydrate. The patternwas generated as described above and in Example 10. The pattern, shownin FIG. 9, exhibited characteristic reflections corresponding tod-spacings as follows.

Relative Position d-spacing Intensity [° 2 θ] [{acute over (Å)} ({acuteover (Å)} = 0.1 nm)] [%] 6.7 13.3 4.9 8.4 10.6 100.0 13.4 6.6 17.9 14.96.0 17.0 15.6 5.7 11.4 16.0 5.5 29.4 17.7 5.0 26.7 19.1 4.7 28.8 19.84.5 16.1 23.7 3.8 45.4 25.5 3.5 25.3

In addition to the X-ray powder diffraction patterns described above,the crystalline IPA solvate/hydrate was further characterized bysolid-state carbon-13 nuclear magnetic resonance (¹³C NMR) spectra. The¹³C NMR spectra were recorded as described above. FIG. 10 shows thesolid state ¹³C CPMAS NMR spectrum for the crystalline IPAsolvate/hydrate. Characteristic peaks are observed at 172.7, 171.4,161.4, 157.7, 156.3, 139.8, 133.3, 131.2, 129.0, 121.4, 104.6, 102.4,74.9, 62.5, 61.7, 59.1, 57.6, 54.8, 52.7, 36.6, 35.2, 29.8, 28.4, 27.2,25.1, 24.8, 19.9, 19.2 and 13.2 ppm.

Further, a differential scanning calorimetry (DSC) curve was prepared asdescribed above. FIG. 11 shows a typical DSC curve for the crystallineIPA solvate/hydrate.

Example 15 Preparation of Macrocyclic Ester

A mixture of the alkyne macrocyclic ester from Example 13 (10.0 g, 17.71mmol) and 5% Pd/C 50% wet (3.5 g, 0.822 mmol) in THF (100 mL) washydrogenated at RT under 40 psig of H₂ for at least 10 h. Upon reactioncompletion, the batch was filtered through CELITE® (filter aid, FisherScientific, Fair Lawn, N.J.), and the filtered catalyst was washed withTHF (100 mL). The combined filtrate was solvent-switched to IPA invacuum at a final volume of ˜50 mL. The slurry was heated up to refluxfor about 1 h. The batch was then cooled to 50° C. and water (30 mL) wasadded dropwise over 1 h. The batch was slowly cooled to below 0° C. over2 h and stirred at 0° C. for additional 1 h before filtration. The wetcake was washed with a cold solution (0° C. to 5° C.) of 57% IPA inwater (17.5 mL). Suction drying at RT gave 8.5 g of the desiredmacrocyclic ester.

¹H NMR (400 MHz, CDCl₃) δ 7.83 (d, J=9.2 Hz, 1 H), 7.18 (dd, J=9.2, 2.8Hz, 1 H), 7.1 (d, J=2.8 Hz, 1 H), 5.98 (t, J=4.0 Hz, 1 H), 5.24 (d,J=9.9 Hz, 1 H), 4.60 (dd, J=10.7, 7.3 Hz, 1 H), 4.46 (d, J=11.9 Hz, 1H), 4.40 (d, J=10.0 Hz, 1 H), 4.01 (dd, J=11.6, 4.0 Hz, 1 H), 3.93 (s, 3H), 3.80 (m, 1 H), 3.75 (s, 3 H), 2.90 (ddd, J=13.7, 11.5, 4.8 Hz, 1 H),2.79 (ddd, J=13.7, 12.1, 4.8 Hz, 1 H), 2.69 (dd, J=14.2, 6.5 Hz, 1 H),2.28 (ddd, J=14.5, 10.7, 4.3 Hz, 1 H), 1.76 (m, 2 H), 1.66 (m, 2 H),1.52 (m, 3 H), 1.09 (s, 9 H), 0.99 (m, 1 H), 0.92 (m, 1 H), 0.67 (m, 1H), 0.46 (m, 1 H).

¹³C NMR (100 MHz, CDCl₃) δ 172.4, 171.5, 160.4, 157.5, 155.1, 148.7,140.1, 134.6, 129.4, 118.7, 106.1, 74.4, 59.4, 58.2, 55.8, 55.5, 54.4,52.5, 35.7, 35.2, 34.0, 30.9, 29.5, 28.6, 28.3, 26.5, 18.9, 11.2.

IPC HPLC conditions: Ascentis Express C18 Column, 100×4.6 mm, 2.7 μm;Column temperature or 40° C.; Flow rate or 1.8 mL/min; and Wavelength of215 nm.

Gradiant: min CH₃CN 0.1% H₃PO₄ 0 10 90 6 95 5 9 95 5 9.1 10 90 Retentiontimes: min. Alkyne macrocyclic ester 5.7 cis-Alkene macrocclic ester(reaction intermediate) 6.0 trans-Alkene macrocclic ester (reactionintermediate) 6.0 Product of Example 15 6.2

Example 16 Preparation of Macrocvclic Acid

To a slurry of macrocyclic ester from Example 14 (90 g, 158.3 mmol) inMeOH (720 mL) at RT was added 2 M NaOH (237.4 mL, 475 mmol) dropwise.The reaction mixture was aged at 50° C. for 2 h to 3 h. The reactionsolution was cooled to 35° C. to 40° C., and 5 N HCl in 50% aq MeOH (70mL) was added dropwise. The batch was seeded with free acid hemihydrate(˜100 mg) and aged for 30 min to 1 h at 40° C. Additional 5 N HCl in 50%aq MeOH (30 mL) was added dropwise over 2 h to 4 h at 40° C. The slurrywas aged additional 1 h before cooling to RT. The slurry was aged foradditional 1 h before filtration. The wet cake was washed with 65% MeOHin water (3×270 mL, displacement wash, slurry wash and displacementwash). Suction drying at RT or vacuum-oven drying with dry N₂ sweep at60° C. to 80° C. gave 85.6 g of macrocyclic acid hemihydrate as anoff-white solid.

¹H NMR (400 MHz, CDCl₃) δ 7.85 (d, J=9.0 Hz, 1 H), 7.19 (dd, J=9.0, 2.8Hz, 1 H), 7.13 (d, J=2.8 Hz, 1 H), 5.99 (t, J=3.9 Hz, 1 H), 5.45 (d,J=9.9 Hz, 1 H), 4.80 (s, br, 2 H, COOH, hemihydrate H₂O), 4.64 (dd,J=10.4, 7.4 Hz, 1 H), 4.49 (d, J=11.6 Hz, 1 H), 4.44 (d, J=10.0 Hz, 1H), 3.99 (dd, J=11.7, 4.0 Hz, 1 H), 3.94 (s, 3 H), 3.81 (m, 1 H), 2.90(ddd, J=13.8, 11.8, 4.8, 1 H), 2.80 (ddd, J=13.8, 11.8, 4.8 Hz, 1 H),2.71 (dd, J=14.3, 7.3, 1 H), 2.42 (ddd, J=14.4, 10.6, 4.2 Hz, 1 H), 1.76(m, 2 H), 1.66 (m, 2 H), 1.52 (m, 3 H), 1.07 (s, 9 H), 0.96 (m, 2 H),0.67 (m, 1 H), 0.47 (m, 1 H).

¹³C NMR (100 MHz, CDCl₃) δ 174.5, 172.1, 160.5, 157.6, 155.1, 148.6,141.0, 134.3, 129.1, 118.9, 106.1, 74.3, 59.6, 58.3, 55.6, 54.6, 35.6,35.3, 33.7, 30.8, 29.4, 28.6, 28.3, 26.5, 18.9, 11.2.

IPC HPLC conditions: Hypersil Gold PFP Column, 150×4.6 mm, 3.0 μm,Column temperature of 40° C.; Flow rate of 1.8 mL/min; and Wavelength of215 nm.

Gradiant: min CH₃CN 0.1% H₃PO₄ 0 25 75 12 80 20 12.1 25 75 14 25 75Retention times: min. Product of Example 15 6.78 Product of Example 165.41

Example 17 Preparation of Compound A, Method A

Macrocyclic acid hemihydrate, the product of Example 15 (10.16 g, 18.03mmol) was dissolved in THF (50 mL to 90 mL). The solution wasazetropically dried at a final volume of 100 mL. Sulfonamide pTSA salt(7.98 g, 1.983 mmol) followed by DMAc (15 mL) was added at RT. The batchwas cooled to 0° C. to 10° C., and pyridine (10 mL) was added dropwise.Then, EDC HCl (4.49 g, 23.44 mmol) was added in portions or one portionat 0° C. to 10° C. The reaction mixture was aged at 0° C. to 10° C. for1 h, and then warmed to 15° C. to 20° C. for 2 h to 4 h. MeOAc (100 mL)followed by 15 wt % citric acid in 5% NaCl in water (50 mL) was added,while the internal temperature was maintained to <25° C. with externalcooling. The separated organic phase was washed with 15 wt % citric acidin 5% NaCl in water (50 mL) followed by 5% NaCl (50 mL). The organicphase was solvent-switched to acetone at a final volume of =80 mL. Water(10 mL) was added dropwise at 35° C. to 40° C. The batch was seeded withCompound A monohydrate form III (=10 mg) and aged for 0.5 h to 1 h at35° C. to 40° C. Additional water (22 mL) was added dropwise over 2 h to4 h at 35° C. to 40° C. The slurry was aged at 20° C. for 2 h to 4 hbefore filtration. The wet cake was displacement washed with 60% acetonein water (2×40 mL). Suction drying at RT gave Compound A monohydrateform III as a white solid.

¹H NMR (400 MHz, CDCl₃) δ 9.95 (s, br, 1 H), 7.81 (d, J=9.1 Hz, 1 H),7.18 (dd, J=9.1, 2.7 Hz, 1 H), 7.16 (s, br, 1 H), 7.13 (d, J=2.7 Hz, 1H), 5.96 (t, J=3.8 Hz, 1 H), 5.72 (m, 1 H), 5.68 (d, J=10.1 Hz, 1 H),5.19 (d, J=17.1 Hz, 1 H), 5.07 (d, J=10.1 Hz, 1 H), 4.52 (d, J=11.4 Hz,1 H), 4.45 (d, J=9.8 Hz, 1 H), 4.36 (d, J=10.5, 6.9 Hz, 1 H), 4.05 (dd,J=11.5, 3.9 Hz, 1 H), 3.93 (s, 3 H), 3.78 (m, 1 H), 2.90 (m, 1 H), 2.82(tt, J=8.0, 4.8 Hz, 1 H), 2.74 (dt, J=13.2, 4.8 Hz, 1 H), 2.59 (dd,J=14.0, 6.7 Hz, 1 H), 2.40 (ddd, J=14.0, 10.6, 4.0 Hz, 1 H), 2.10 (dd,J=17.7, 8.7 Hz, 1 H), 1.98 (2 H, mono hydrate H₂O), 1.88 (dd, J 8.2, 5.9Hz, 1 H0, 1.74 (m, 3 H), 1.61 (m, 1 H), 1.50 (m, 3 H), 1.42 (dd, J=9.6,5.8 Hz, 1 H), 1.22 (m, 2 H), 1.07 (s, 9 H), 0.95 (m, 4 H), 0.69 (m, 1H), 0.47 (m, 1 H).

¹³C NMR (100 MHz, CDCl₃) δ 173.5, 172.1, 169.1, 160.4, 157.7, 154.9,148.4, 141.0, 134.3, 132.7, 129.1, 118.8, 118.7, 106.5, 74.4, 59.6,59.4, 55.8, 55.5, 54.9, 41.8, 35.4, 35.3, 35.2, 34.3, 31.2, 30.7, 29.5,28.6, 28.2, 26.6, 22.6, 18.7, 11.2, 6.31, 6.17.

HPLC conditions: Ascentis Express Column, 10 cm×4.6 mm, 2.7 μm; Columntemperature of 40° C.; Flow rate of 1.8 mL/min; and Wavelength of 215nm.

Gradiant: min CH₃CN 0.1% H₃PO₄ 0 20 80 5 55 45 15 55 45 25 95 5 27 95 527.1 20 80 32 20 80 Retention time: min. Compound A 14.50

Example 18 Preparation of Compound A, Method B

To a 50-L flask equipped with overhead stirring was added macrocyclicacid (1.06 kg crude, 1.00 eq), amine-pTSA (862 g crude, 1.12 eq) andMeCN (7.42 L) at 19° C. The slurry was cooled in a water bath, pyridine(2.12 L, 13.8 eq) was added, aged 15 min, and then added EDC (586 g,1.60 eq) in one portion, aged 1.5 h, while it turned into a clearhomogeneous solution.

The solution cooled in a water bath, then quenched with 2 N HCl (1.7 L),and seeded (9.2 g), aged 15 min, and the rest of the aqueous HCl wasadded over 2.5 h. A yellow slurry was formed. The slurry was agedovernight at RT, filtered, washed with MeCN/water (1:1 v/v, 8 L), toobtain Compound A (Hydrate II).

Compound A was dissolved in acetone (4 L) at RT, filtered andtransferred to a 12-L round-bottom flask with overhead stirring, rinsedwith extra acetone (1 L), heated to 50° C., water (0.9 L) was added,seeded with Compound A monohydrate form III (˜10 mg), and aged 15 min,and then added water (0.8 L) over 2.5 h, extra water 3.3 v over 2.5 hwas added, stopped heating, cooled to RT, aged at RT overnight,filtered, washed with water/acetone (1:1 v/v, 4 L), and dried in airunder vacuum. Compound A Hydrate III, 670 g, was obtained as anoff-white solid.

Example 19 Preparation of Compound A, Method C

Macrocyclic acid hemihydrate from Example 15 (10.16 g, 18.03 mmol) wasdissolved in THF (50 ml to 90 mL). The solution was azetropically driedat a final volume of 100 mL. Sulfonamide pTSA salt (7.98 g, 19.83 mmol)was added, followed by DMAc (15 mL), at RT. The batch was cooled to 0°to 10° C., and pyridine (10 mL) was added dropwise. Then, EDC HCl (4.49g, 23.44 mmol) was added (in portions or one portion) at 0° C. to 10° C.The reaction mixture was aged at 0° C. to 10° C. for 1 h, and thenwarmed to 15° C. to 20° C. for 2 h to 4 h. THF (50 mL) was added,followed by 15 wt % citric acid in 15 wt % aq. NaCl (50 mL), while theinternal temperature was maintained at <25° C. with external cooling.The separated organic phase was washed with 15 wt % citric acid in 15%aq. NaCl (40 mL), followed by 15% NaCl (40 mL). The organic phase wassolvent-switched to acetone at a final volume of =75 mL. Water (11 mL to12 mL) was added dropwise at 35° C. to 40° C. The batch was seeded withCompound A monohydrate form III (˜20 mg) and aged for 0.5 h to 1 h at35° C. to 40° C. Additional water (22 mL) was added dropwise over 2 h to4 h at 35° C. to 40° C. The slurry was aged at 20° C. for 2 h to 4 hbefore filtration. The wet cake was displacement washed with 60% acetonein water (40 mL×2). Suction drying at RT or vacuum-oven drying at 45° C.gave Compound A monohydrate form III as a white solid.

Example 20 Preparation of Compound A, Method D

Macrocyclic acid hemihydrate from Example 12 (10 g, 98.4 wt %, 17.74mmol) was dissolved in THF (70 mL). The solution was azetropically driedat a final volume of ˜60 mL. Sulfonamide pTSA salt (7.53 g, 18.7 mmol)was added at RT. The batch was cooled to 0° C. to 5° C., and pyridine(11.4 mL) was added dropwise. Then, EDC HCl (4.26 g, 22.2 mmol) wasadded in portions at 0° C. to 15° C. The reaction mixture was aged at10° C. to 15° C. for 2 h to 4 h. 35 wt % Citric acid in 10 wt % aq. NaCl(80 mL) was added, while the internal temperature was maintained at <25°C. with external cooling. The separated organic phase wassolvent-switched to acetone at a final volume of ˜75 mL. Water (12 mL)was added dropwise at 50° C. The batch was seeded with Compound Amonohydrate form III (˜300 mg) and aged for 0.5 h to 1 h at 50° C.Additional water (25 mL) was added dropwise over 6 h at 35° C. to 40° C.The slurry was aged at 20° C. for 2 h to 4 h before filtration. The wetcake was displacement washed with 65% acetone in water (40 mL). Suctiondrying at RT or vacuum-oven drying at 45° C. gave Compound A monohydrateform III as a white solid.

It will be appreciated that various of the above-discussed and otherfeatures and functions, or alternatives thereof, may be desirablycombined into many other different systems or applications. Also thatvarious presently unforeseen or unanticipated alternatives,modifications, variations or improvements therein may be subsequentlymade by those skilled in the art which are also intended to beencompassed by the following claims.

What is claimed is:
 1. A method of making compounds of Formula C:

wherein n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6,7 and 8; X¹ and X² are each independently selected from the groupconsisting of Br, Cl and I; and R⁵ is CF₃; said method comprising: (1)reacting

 where LG is selected from the group consisting of halogen atoms,—O—SO₂R⁸, —O—PO(OR⁸)₂ and a protecting group and each R⁸ isindependently selected from the group consisting of C₁₋₈ alkyl, C₃₋₈cycloalkyl, aryl and heteroaryl groups and each R⁸ is independentlysubstituted by 0, 1, 2, 3 or 4 substituents independently selected fromthe group consisting of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl,halogen, —NH₂ and —OH, and the protecting group is selected from —OSiR⁸and —OR⁸, with a chiral alcohol and

 to produce

 where each R¹ is independently selected from the group consisting ofC₁₋₈ alkyl, aryl and heteroaryl groups, or two R¹ are taken, togetherwith the O—P—O atoms to which they are attached, to form a ringcontaining 5-19 atoms; and where R² and R³ are each selected from thegroup consisting of H, C₁₋₈ alkyl and —O—C₁₋₈ alkyl groups, or R² and R³are taken together with the nitrogen atom to which they are attached toform a ring containing 5-19 atoms; (2) reacting

 with a Grignard reagent to produce

 where R⁴ is selected from the group consisting of C₁₋₈ alkyl, aryl, andheteroaryl groups and R⁴ is substituted by 0, 1, 2, 3 or 4 substituentsindependently selected from the group consisting of C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, aryl, halogen, —NH₂ or —OH; (3) halogenating

 to produce

 where X¹ and X² are each independently selected from the groupconsisting of Br, Cl and I; and (4) reacting

 with triflouroacetic anhydride to produce

 where R⁵ is CF₃.
 2. A method of making a compound of Formula B:

or a salt thereof, wherein n is selected from the group consisting of 0,1, 2, 3, 4, 5, 6, 7 and 8; and R⁷ is selected from the group consistingof acetyl and

and R⁶ is selected from the group consisting of C₁₋₈ alkyl, C₃₋₈cycloalkyl, aryl, and heterocycle groups and R⁶ is substituted by 0, 1,2, 3 or 4 substituents independently selected from the group consistingof C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl, halogen, —NH₂ and —OH,and salts thereof; said method comprising: preparing a compound ofFormula C according to the method of claim 1; and converting

 or a salt thereof.
 3. The method of claim 1, wherein

in step (1) is prepared by: (i) reacting

 where n is as defined in claim 1 and X is a halogen atom, with amagnesium source to produce

and (ii) reacting

 with

 to produce

 where LG is defined as in claim
 1. 4. The method of claim 3, wherein Xis Br.
 5. The method of claim 2, wherein said converting

 comprises: (i) de-halogenating

 to produce

(ii) reacting

 with a reagent containing a leaving group to produce

 where LG is said leaving group and is selected from the groupconsisting of halogen atoms, —O —SO₂R⁸, —O —PO(OR⁸)₂and a protectinggroup and each R⁸is independently selected from the group consisting ofC₁₋₈alkyl, C₃₋₈cycloalkyl, aryl, and heteroaryl groups and each R⁸isindependently substituted by 0, 1, 2, 3,or 4 substituents independentlyselected from the group consisting of C₁₋₆alky, C₂₋₆alkenyl,C₂₋₆alkynyl, aryl halogen, —NH₂, and —OH, and the protecting group isslected from —OSiR⁸ and —OR⁸; (iii) reacting

 with

 to produce

 wherein R⁷ is

and (iv) optionally forming a salt of


6. The method of claim 2, wherein R⁶ is selected from the groupconsisting of C₂C₆ alykl groups.
 7. The method of claim 2, wherein R⁶ istert-butyl.
 8. The method of claim 1, wherein LG is Cl.
 9. The method ofclaim 1, wherein, in step (1), the chiral alcohol is chlorohydrin. 10.The method of claim 1, wherein, in step (1),


11. The method of claim 5, wherein step (iv) comprises forming a salt ofthe compound, wherein the salt is selected from tert-butylamine salt,dibenzylamine salt, and dicyclohexyl amine salt.
 12. The method of claim2, wherein the compound of Formula B has the following structuralformula:

or a salt thereof, said method comprising: (1) reacting

 with chlorohydrin and

 to produce

(2) reacting

 with a Grignard reagent to produce

(3) brominating

 to produce

(4) reacting

 with trifluoroacetic anhydride to produce

(5) converting

(6) optionally converting

 to a salt thereof.
 13. A method of preparing Compound A:

or a pharmaceutically acceptable salt thereof, said method comprising:(a) producing a compound of Formula B according to claim 12, (b)reacting

 with

 to produce

 where PG is H or a protecting group selected from —OSiR⁸ and —OR⁸; (c)and further comprising the steps of: